NO319755B1 - Heterocyclic Inhibitors of P38 - Google Patents

Description

The present invention relates, as defined in claim 1, to inhibitors of p38, a mammalian protein kinase involved in cell proliferation, cell death and response to extracellular stimuli. The invention also relates to methods for producing these inhibitors. The invention also provides pharmaceutical compositions comprising the inhibitors according to the invention and methods for utilizing these compositions in the treatment and prevention of various disorders. Finally, the present invention encompasses the use of compounds and compositions according to the invention for the preparation of a drug to treat or prevent a disease or condition associated with p38 activation.

The invention background

Protein kinases are involved in various cellular responses to extracellular signals. Recently, a family of mitogen-activated protein kinases (MAPKs) has been discovered. Members of this family are Ser/Thr kinases that activate their substrates by phosphorylation [B. Stein et al., Ann. Rep. With. Chem., 31, pp. 289-98 (1996)]. MAPKs are themselves activated by several different signals including growth factors, cytokines, UV irradiation, and stress-inducing agents.

A particularly interesting MAPK is p38. p38, also known as cytokine suppressive anti-inflammatory drug-binding protein (CSBP) and RK, was isolated from murine pre-B cells transfected with the lipopolysaccharide (LPS) receptor, CD14, and induced with LPS. P38 has since been isolated and sequenced, as has the cDNA encoding it in humans and mice. Activation of p38 has been observed in cells stimulated by stress, such as lipopolysaccharide (LPS) treatment, UV, anisomycin, or osmotic shock, and by cytokines, such as IL-1 and TNF.

Inhibition of p38-kinase leads to a block on the production of both IL-1 and TNF. IL-1 and TNF stimulate the production of other pro-inflammatory cytokines such as IL-6 and IL-8 and have been implicated in acute and chronic inflammatory diseases and in post-menopausal osteoporosis [R. B. Kimble et al., Endocrinol., 136, pp. 3054-61 (1995)].

Based on these findings, it is hypothesized that p38, together with other MAPKs, plays a role in mediating cellular responses to inflammatory stimuli, such as leukocyte accumulation, macrophage/monocyte activation, tissue resorption, fever, acute phase responses and neutrophilia. In addition, MAPKs, such as p38, have been implicated in cancer, thrombin-induced platelet aggregation, immunodeficiency disorders, autoimmune diseases, cell death, allergies, osteoporosis, and neurodegenerative disorders. Inhibitors of p38 have also been implicated in the field of pain management through inhibition of prostaglandin endoperoxide synthase-2 induction. Other diseases associated with overproduction of IL-1, IL-6, IL-8 or TNF are shown in WO 96/21654.

Others have already begun to try and develop drugs that specifically inhibit MAPKs. For example, PCT publication WO 95/31451 describes pyrazole compounds that inhibit MAPKs, and, in particular, p38. However, the activity of these inhibitors in vivo is still being investigated.

Accordingly, there is still a great need to develop other potent inhibitors of p38, including p38-specific inhibitors, which are useful in the treatment of various conditions associated with p38 activation.

Summary of the invention

The present invention addresses these problems by providing compounds that exhibit strong inhibition of p38.

These compounds have the general formulas:

wherein each Q 1 and Q 2 is independently selected from phenyl or naphthyl; the rings constituting Qi are substituted with 1 to 4 substituents each of which is independently selected from halo; C1-C3-alkyl or O-(C1-C3)-alkyl; the rings constituting Q2 are optionally substituted with up to 4 substituents, each of which is independently selected from halo; straight or branched C 1 -C 3 alkyl optionally substituted with OH; R' is selected from hydrogen; (C 1 -C 3 )-alkyl; phenyl or phenyl substituted with 1 to 3 substituents independently selected from halo, methoxy, methyl or ethyl; or a 5-6 membered heterocyclic ring system optionally substituted with 1-3 substituents independently selected from halo, methoxy, hydroxy, methyl or ethyl, wherein the heterocyclic ring system is selected from pyridyl, morpholinyl, piperazinyl, dihydrothiazolyl, tetrazolyl, piperidinyl, triazolyl or pyrrolidinyl ; R3 is selected from a 5-8 membered aromatic or non-aromatic carbocyclic or heterocyclic ring system, each optionally substituted with R', R<4> or -C(0)R', wherein the ring system is selected from phenyl, cyclohexyl, piperazinyl, pyrrolidinyl, piperidinyl, homopiperazinyl, morpholinyl, imidazolyl or triazolyl; R<4> is straight or branched (C1-C4) alkyl optionally substituted with OR', CO2R<r> or CON(R')2; or a 5-6 membered carbocyclic or heterocyclic ring system; R<5> is selected from hydrogen; (C 1 -C 3 )-alkyl, pyridinyl, morpholinyl, or tetrazolyl; W is selected from H; N(R2)C(O)-OR2; N(R2)C(O)-N(R2)2; N(R<2>)C(O)-N(R2)(R<3>); N(R2)C(O)-R2; N(R<2>)2; C(O)-R 2 ; C(O)-N(R 2 ) 2 ; C (0)-OR<2>; J; or straight chain or branched (C1-C4)-alkyl optionally substituted with N(R')2, OR', CO2R', CON(R')2, R<3>, SO2N(R<2>)2, 0C( 0)R<2>, 0C(0)R', OC(0)N(R<2>)2, -N(R<4>)(R<5>), -C (O) N (R5 ) (R2) , -N (R2) C (0) N (R2) (R5) , -NC(0)0R<5>, -OC(0)N (R2) (R5) , -J; NHS02-R2, NHS02-R<3>, or 0P03H2; provided that if U is W, W is not R<3->substituted C 1 -alkyl; each R is independently selected from hydrogen, -R<2>, -N{R<2>)2 or -OR<2>; R<2> is selected from hydrogen or (C1-C3)-alkyl, optionally substituted with -N(R')2, -OR', -C (0)-N (R'} 2, -C(0) -OR', -C(O)N=C(NH)2 or R<3>;

Y is C;

Z is CH or N;

U is selected from R or W;

V is -C(O)NH 2 ;

A, B and C are independently selected from -O-, -NH- or -CH2-

J is a straight or branched (C 1 -C 4 )alkyl derivative substituted with 1-3 substituents selected from D, -T-C{O)R', or -PO 3 H 2 ;

D is selected from the group

T is either 0 or NH; and

G is either NH2 or OH,

provided that in compounds of formula (Ia) where W is hydrogen, then U is not hydrogen, and

in compounds of formula (Ia) where W is hydrogen and U is -R<2>, -N(R<2>)2 or -OR2, then each of these R<2> is not hydrogen, (C1-C3) -alkyl or (C1-C3) -alkyl substituted by -N(R')2, -OR', -C(0)-N(R')2, -C(0)-0R' or an unsubstituted 5- 6-membered aromatic carbocyclic or heterocyclic ring system.

In another embodiment, the invention provides pharmaceutical compositions comprising the p38 inhibitors according to the present invention. These compositions can be used in methods for treating or preventing several different disorders, such as cancer, inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative diseases, infectious diseases, viral diseases and neurodegenerative diseases. These compositions are also applicable in methods to prevent cell death and hyperplasia and can therefore be used to treat or prevent reperfusion/ischemia in stroke, heart attack, and organ hypoxia. The compositions are also useful in methods of preventing thrombin-induced platelet aggregation. Each of these methods described above is also part of the present invention.

The present invention also relates to the use of a compound or composition according to the invention for the production of a drug to treat or prevent diseases or conditions associated with p38 activation as mentioned above.

According to a preferred embodiment, Qi is selected from phenyl or containing 1 to 3 substituents, wherein at least one of said substituents is in the ortho position and said substituents are independently selected from chlorine, fluorine, bromine, -CH3, -OCH3 or -O( CH 2 ) 2 CH 3 .

Even more preferred is phenyl containing at least 2 of the above-indicated substituents, both in the ortho position.

Some specific examples of preferred Qi are:

Most preferably, Ch is selected from 2,6-difluorophenyl or 2,6-dichlorophenyl.

According to a preferred embodiment, Q 2 is phenyl or naphthyl containing 0 to 3 substituents, wherein each substituent is independently selected from chlorine, fluorine, bromine, methyl, ethyl, isopropyl and -CH 2 OH.

Some specific examples of preferred Q2 are:

or unsubstituted phenyl.

Most preferred are compounds in which Q2 is selected from phenyl, 2-isopropylphenyl, 3,4-dimethylphenyl, 2-ethylphenyl, 3-fluorophenyl, 2-methylphenyl, 3-chloro-4-fluorophenyl, 3-chlorophenyl, 2-methyl-4 -chlorophenyl, 2-bromophenyl, 2-methylenehydroxyphenyl, 4-fluorophenyl, 2-methyl-4-fluorophenyl, 2-chloro-4-fluorophenyl, 2,4-difluorophenyl, 2-methylenehydroxy-4-fluorophenyl, 1-naphthyl, 3 - chloro-2-methylenehydroxy, 3-chloro-2-methyl, or 4-fluoro-2-methyl.

According to an even more preferred embodiment, each Y C and R and U bonded to each Y component is selected from hydrogen or methyl.

According to another preferred embodiment, W is a 0-4 atom chain terminating in an alcohol, amine, carboxylic acid, ester, amide or heterocycle.

Some specific examples of preferred W are:

Most preferably, W is selected from:

U has the same preferred and most preferred embodiments as W.

According to an even more preferred embodiment, each Y is C, and W and/or U is not hydrogen.

Some preferred embodiments are given in Table 1 below:

Particularly preferred embodiments include:

and X is selected from H, or

Particularly preferred embodiments also include and X is selected from NH 2 or N(CH 3 ) 2 ,

wherein X is OH, NH 2 , or N(CH 3 ) 2 .

Other particularly preferred embodiments include:

Other particularly preferred embodiments include:

Other particularly preferred embodiments include:

Most preferred embodiments include:

According to another embodiment, the present invention provides methods for preparing above-identified inhibitors of p38 of formulas (Ia) and (Ic). Representative synthesis schemes for formula (Ia) are illustrated below.

Schemes 1-3 illustrate the preparation of compounds in which W is either an amino, carboxyl or an aldehyde function. In each case, the particular unit can be modified through chemistry well known in the literature. For example, the last amino compounds D and N (Schemes 1 and 4, respectively) can be acylated, sulfonylated or alkylated to produce compounds within the scope of W. In all schemes, the L1 and L2 groups on the starting materials are intended to represent leaving groups ortho to the nitrogen atom in a heterocyclic ring. For example, compound A may be 2,6-dichloro-3-nitropyridine.

In reaction scheme 1, W is selected from amino-derivatized compounds such as N (R 2 ) C (O)-OR 2 ; N(R2)C(O)-N(R2)2; N(R2)C(0)-N(R2)(R3); N(R2)C(O)-R2; or N(R<2>)2.

In Reaction Scheme 1, the Q2 ring is introduced using one of many reactions known in the art that result in the production of biaryl compounds. An example would be the reaction of an aryllithium compound with the pyridine intermediate A. Alternatively, an arylmetallic compound such as an arylstannane or an arylboronic acid can be reacted with the aryl halide moiety (intermediate A) in the presence of a Pd° catalyst to form product B. In the next step, a Qi-substituted derivative such as phenylacetonitrile derivative can be treated with a base such as sodium hydride, sodium amide, LDA, lithium hexamethyldisilazide, or any number of other non-nucleophilic bases to deprotonate the alpha position of the cyano group, which represents a masked amide unit. This anion is then brought into contact with intermediate B to form C. The nitrile group or equivalent group of intermediate C is then hydrolyzed to form the amide, and the nitro group is subjected to reducing conditions to form the amine intermediate D. Intermediate D is then used to introduce different functionality defined by W through chemistry such as acylation, sulfonylation, or alkylation reactions well known in the literature. Depending on the regiochemistry of the first two steps of this procedure, it may be necessary to reverse the first two steps.

In reaction scheme 2, W is selected from carboxyl-derivatized compounds such as C(O)-R2; C(O)-N(R 2 ) 2 ; or C(O) -OR 2 .

Reaction core 2 generally follows the procedures described for reaction core 1 except that a carboxyl intermediate such as E is the starting material. The first two steps mirror Scheme 1 and, as mentioned for Scheme 1, can be reversed depending on the regiochemistry of specific examples. Intermediate G formed from these first two steps and this material can be hydrolyzed as mentioned for the carboxyl intermediate H. The carboxyl group can then be modified according to well-known procedures from the literature to prepare analogues with defined W substituents such as acylations, amidations and esterifications.

In reaction scheme 3, W is selected from linear or branched {C1-C4)-alkyl optionally substituted with N(R')2, OR', CO2R', CON (R')2, R3 or SO2N(R<2>)2 provided that W is not an R<3->substituted C 1 -alkyl.

In reaction scheme 3, a pyridine derivative is metallated and stopped with one of many known electrophiles that can generate an aldehyde, to form intermediate I. The aldehyde can be masked to form the dimethyl acetal J. This intermediate is then proceeded as described in reaction cores 1 and 2 to introducing the Q1 and Q2 substituents, to produce intermediate L. As before, these two steps can be interchanged depending on the specific regio-chemistry. The masked aldehyde of L can then be deprotected and used to form compounds with the defined W substitution by using well-known chemistry such as alkylations and reductive aminations.

Reaction schemes 4-6 are similar to reaction schemes 1-3 except that the target compounds are those in which Z = nitrogen. The steps for these reaction schemes are parallel to 1-3 except that the alkylation using a phenylacetonitrile is replaced by a reaction with a Q1-amine derivative such as a substituted aniline derivative. The amide of the molecule is then introduced into an acylation reaction with, for example, chlorosulfonyl isocyanate.

In reaction scheme 4, W is selected from amino-derivatized groups such as N (R 2 ) C (O)-OR 2 ; N(R2)C(O)-N(R2)2; N(R<2>)C(O)-N(R2)(R<3>); N(R2)C(O)-R2; or N(R<2>)2.

In reaction scheme 4, intermediate B (from reaction scheme 1) is treated with, for example, an aniline derivative in the presence of a base such as potassium carbonate. In addition, a palladium catalyst can be used to enhance the reactivity of this general reaction type, if necessary. The resulting amine derivative is then acylated to form intermediate M. The nitro group in M is then reduced to form N and the amino group can then be derivatized as described for Reaction Scheme 1. As mentioned for Reaction Schemes 1-3, the steps involved in the introduction of QI- and The Q2 substituents are interchanged depending on the specific regiochemistry of specific compounds.

In reaction scheme 5, W is selected from carboxyl derivatized groups such as C(0)-R<2>; C(O)-N(R2)2; or C(0)-OR<2>.

In reaction scheme 6, W is selected from linear or branched (C1-C4)-alkyl optionally substituted with N(R')2, OR', CO2R', CON (R')2, R<3>, or SO2N(R< 2>)2 provided that W is not an R<3->substituted C 1 alkyl.

Reaction schemes 5 and 6 generally follow the procedures mentioned above.

Those skilled in the art will recognize that reaction schemes 1-6 can be used to synthesize compounds having the general formula (Ic).

According to another embodiment of the invention, the activity of p38 inhibitors according to the present invention can be analyzed in vitro, in vivo or in a cell line. In vitro assays include assays that determine inhibition of either the kinase activity or the ATPase activity of activated p38. Alternative in vitro assays quantify the ability of the inhibitor to bind to p38, and this can be measured by radiolabeling the inhibitor before binding, isolating the inhibitor/p38 complex and determining the amount of bound radiolabeled material or by running a competition experiment where new inhibitors are incubated with p38 bound to known radioligands.

Cell culture assays of the inhibitory effect of the compounds of the present invention can determine the amounts of TNF, IL-1, IL-6 or IL-8 produced in whole blood or cell fractions thereof in cells treated with inhibitor compared to cells treated with negative controls. Levels of these cytokines can be determined through the use of commercially available ELISAs.

An in vivo assay that can be used to determine the inhibitory activity of the p38 inhibitors of the present invention is the suppression of hindpaw edema in rats with Afycojbacterium þutyricujn-induced adjuvant arthritis. This is described in J.C. Boehm et al., J. Med. Chem. 39, pp. 3929-37 {1996}, the presentations therein are hereby incorporated by reference. The p38 inhibitors according to the present invention can also be analyzed in animal models for arthritis, bone resorption, endotoxin shock and immune function, as described in A. M. Badger et al., J. Pharmol. Experimental Therapeutics, 279, pp. 1453-61 (1996), the presentations herein are hereby incorporated by reference.

The P38 inhibitors or pharmaceutical salts thereof can be formulated into pharmaceutical compositions for administration to animals or humans. These pharmaceutical compositions, comprising an amount of p38 inhibitor effective to treat or prevent a p38-mediated condition or a pharmaceutically acceptable carrier, are another embodiment of the present invention.

The term "p38-mediated condition", as used herein, means any disease or other harmful condition in which p38 is known to play a role. This includes conditions known to be caused by overproduction of IL-1, TNF, IL-6 or IL-8. Such conditions include, without limitation, inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative disorders, infectious disorders, neurodegenerative disorders, allergies, reperfusion/ischemia in stroke, heart attack, angiogenic disorders, organ hypoxia, vascular hyperplasia, cardiac hypertrophy, thrombin-induced platelet aggregation, and conditions associated with prostaglandin endoperoxidase synthase-2.

Inflammatory diseases that can be treated or prevented by the compound of the present invention include, but are not limited to, acute pancreatitis, chronic pancreatitis, asthma, allergies and respiratory distress syndrome in adults.

Autoimmune diseases that can be treated or prevented by the compounds of the present invention include, but are not limited to, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia, autoimmune neutropenia, thrombo -cytopenia, atopic dermatitis, chronic active hepatitis, myasthenia gravis, multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, psoriasis or graft-versus-host reaction.

Destructive bone disorders that can be treated or prevented by the compounds of the present invention include, but are not limited to, osteoporosis, osteoarthritis, and multiple myeloma-related bone disorder.

Proliferative diseases that can be treated or prevented by the compounds of the present invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, and multiple myeloma.

Angiogenic disorders that can be treated or prevented by the compounds of the present invention include solid tumors, ocular neovascularization, infantile hemangioma.

Infectious diseases that can be treated or prevented by the compounds of the present invention include, but are not limited to, sepsis, septic shock, and shigellosis.

Viral diseases that can be treated or prevented by the compounds of the present invention include, but are not limited to, acute hepatitis infection (including hepatitis A, hepatitis B and hepatitis C), HIV infection and CMV retinitis.

Neurodegenerative diseases that can be treated or prevented by the compounds of the present invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia or neurodegenerative diseases caused by traumatic injury.

"p38-mediated conditions" also include ischemia/reperfusion in stroke, heart attack, myocardial ischemia, organ hypoxia, vascular hyperplasia, cardiac hypertrophy, and thrombin-induced platelet aggregation.

In addition, p38 inhibitors according to the present invention are also capable of inhibiting the expression of inducible pro-inflammatory proteins such as prostaglandin endoperoxide synthase-2 (PGHS-2), also referred to as cyclooxygenase-2 {COX-2). Therefore, other "p38-mediated conditions" treatable by the compounds of the invention include edema, analgesia, fever and pain, such as neuromuscular pain, headache, cancer pain, dental pain and pain associated with arthritis.

The diseases that can be treated or prevented by the p38 inhibitors according to the present invention can also be suitably grouped based on the cytokine (IL-1, TNF, IL-6, IL-8) which is believed to be responsible for the disease.

Therefore, an IL-1 mediated disease or condition includes rheumatoid arthritis, osteoarthritis, stroke, endotoxemia, and/or toxic shock syndrome, inflammatory reaction induced by endotoxin, inflammatory bowel disease, tuberculosis, arteriosclerosis, muscular degeneration, cachexia, psoriatic arthritis, Reiter's syndrome , gout, traumatic arthritis, rubella arthritis, acute synovitis, diabetes, pancreatic p-cell disease and Alzheimer's disease.

TNF-mediated disease or condition includes, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, sepsis, septic shock, endotoxic shock, gram-negative sepsis, toxic shock syndrome, acute respiratory distress syndrome in adults, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoidosis, bone resorption disorders, reperfusion injury, graft-versus-host reaction, allograft reactions, fever and myalgias due to infection, cachexia secondary to infection, AIDS, ARC or malignancy, keloid formation, scar tissue formation, Crohn's disease , ulcerative colitis or pyresis. TNF-mediated diseases also include viral infections, such as HIV, SMV, influenza and herpes; and veterinary viral infections, such as lentivirus infections, including but not limited to equine infectious anemia virus, caprine arthritis virus, visnavirus, or maedivirus; or retroviral infections, including feline immunodeficiency virus, bovine immunodeficiency virus, or canine immunodeficiency virus. IL-8-mediated diseases or conditions include diseases characterized by massive neutrophil infiltration, such as psoriasis, inflammatory bowel disease and asthma, cardiac and renal reperfusion injury, acute respiratory distress syndrome in adults, thrombosis and glomerulonephritis.

In addition, the compounds according to the present invention can be used topically to treat or prevent conditions caused or aggravated by IL-1 and TNF. Such conditions include inflamed joints, eczema, psoriasis, inflammatory skin conditions such as sunburn, inflammatory eye conditions such as conjunctivitis, pyrexia, pain and other conditions associated with inflammation.

In addition to the compounds of this invention, pharmaceutically acceptable salts of the compounds of the invention may also be used in compositions to treat or prevent the disorders identified above. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate , hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate , salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, although it is not pharmaceutically acceptable in itself, can be used in the preparation of salts useful as intermediates to obtain the compounds according to the invention and their pharmaceutically acceptable acid addition salts. Salts derived from suitable bases include alkali metal {e.g. sodium and potassium), alkaline earth metal (e.g. magnesium), ammonium and N-{Ci_4-alkyl)4+ salts. The present invention also contemplates the quaternization of any basic nitrogen-containing group in the compounds disclosed herein. Water or oil-soluble products or dispersible products can be obtained by such quaternization.

Pharmaceutically acceptable carriers which can be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate , partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl-pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxyl-methylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool fat.

The compositions according to the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synoampullary, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously.

Sterile injectable forms of the compositions according to the present invention can be aqueous or oily suspensions. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently used as a solvent or suspending agent. For this purpose, any smooth fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectable preparations, and so are naturally pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifiers or bioavailability promoters that are commonly used in the preparation of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for formulation purposes.

The pharmaceutical compositions of the present invention may be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring substances can also be added.

Alternatively, the pharmaceutical compositions according to the present invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agents with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions according to the present invention can also be administered topically, especially when the target of the treatment includes areas or organs that are easily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal system. Appropriate topical formulations are easily prepared for each of these areas and organs.

Topical application for the lower intestinal system can be carried out in a rectal suppository formulation (see above) or in a suitable enema formulation. Topical transdermal patches can also be used.

For topical application, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, paraffin oil, white petroleum jelly, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH-adjusted sterile physiological saline solution, or preferably, as solutions in isotonic, pH-adjusted sterile physiological saline solution, either with or without a preservative such as benzylalkonium chloride. Alternatively, the pharmaceutical compositions may be formulated into an ointment such as petroleum jelly for ophthalmic applications.

The pharmaceutical compositions according to the present invention can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the pharmaceutical formulation art and may be prepared as solutions in physiological saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

The amount of p38 inhibitor that can be combined with the carrier materials to provide a single dosage form will vary depending on the host being treated, the particular mode of administration. Preferably, the compositions should be formulated so that a dosage of between 0.01-100 mg/kg body weight/day of the inhibitor can be administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatment regimen for any specific patient will depend on several different factors, including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, the rate of ringing, drug combination, and the treating physician's assessment and severity of the particular disease to be treated. The amount of inhibitor will also depend on the particular compound in the composition.

The compounds according to the invention can be used for the production of a drug to treat or prevent a condition selected from inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative disorders, infectious diseases, degenerative diseases, allergies, reperfusion/ischemia in stroke, heart attack , angiogenic disturbances, organ hypoxia, vascular hyperplasia, cardiac hypertrophy and thrombin-induced platelet aggregation.

According to another embodiment, the inhibitors according to the present invention are used to treat or prevent a disease or condition mediated by IL-1, IL-6, IL-8 or TNF. Such conditions are described above.

Depending on the specific p38-mediated condition to be treated or prevented, additional drugs commonly administered to treat or prevent that condition may be co-administered with the inhibitors of the present invention. For example, chemotherapeutic agents or other anti-proliferative agents can be combined with the p38 inhibitors of the present invention to treat proliferative diseases.

The additional agents may be administered separately from the p38 inhibitor-containing composition as part of a multiple dosage regimen. Alternatively, these agents may be part of a single dosage form, admixed with the p38 inhibitor in a single composition.

In order that the invention described herein may be more fully understood, the following examples are presented. It should be understood that these examples are for illustrative purposes only and should not be considered as limiting the invention in any way.

EXAMPLE 1

Synthesis of p38 inhibitor compound 6

To a solution of LDA (60 mmol, 40 mL) at -78 °C, was added dropwise a solution of 2,6-dibromopyridine (40 mmol, 9.48 g) in THF (30 mL, dried). The mixture was stirred at -78°C for 20 minutes. Ethyl formate (400 mmol, 32.3 mL) was added and stirring continued at -78 °C for 2 h. Saturated ammonium chloride (200 mL) was added and the mixture warmed to room temperature. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed with aqueous acid and base. The organic phase was dried and evaporated in vacuo. The resulting material was purified by flash chromatography on silica gel, followed by elution with 10% ethyl acetate in n-hexane to give 1 (32 mmol, 8.41 g) as a white solid.

A solution of 1 (13.08 mmol, 3.1 g) and concentrated sulfuric acid (1 mL) in methanol (50 mL) was refluxed overnight. The reaction mixture was cooled, neutralized with aqueous base and extracted into ethyl acetate. Drying and evaporation of the organic phase gave 2 (11.77 mmol, 3.63 g) as an oil.

A solution of t-butoxide (2.2 mmol, 2 mL) was added dropwise to a solution of 2,6-dichloroaniline (1.0 mmol, 162 mg) in THF (2 mL, dried). The mixture was stirred at room temperature for 20 minutes. A solution of 2 (1.0 mmol, 309 mg) in THF (5 mL) was added and stirring continued for 3 h. The reaction mixture was diluted with ethyl acetate and the organic phase was washed with aqueous acid and base. The organic phase was dried and evaporated in vacuo. The resulting material was purified by flash chromatography on silica gel followed by elution with 5% acetone in n-hexane to give 3 (0.33 mmol, 128 mg) as an orange solid.

o -Tolylboronic acid (0.34 mmol, 46 mg), and 3 (0.20 mmol, 80 mg) were dissolved in a toluene/ethanol (5/1) mixture. Thallium-

carbonate (0.5 mmol, 235 mg) and tetrakis(triphenylphosphine)-palladium (0) (10 mg) were added to the solution and the slurry was allowed to reflux for 30 min. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed with aqueous acid and base. The organic phase was dried and evaporated in vacuo. The resulting material was purified by flash chromatography on silica gel followed by elution with 5% methanol in methylene chloride to give 4 (0.17 mmol, 61 mg) as a white solid.

A solution of 4 (0.17 mmol, 61 mg) and chlorosulfonyl isocyanate (1 mmol, 141.5 mg) in methylene chloride (5 mL) was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed with aqueous acid and base. The organic phase was dried and evaporated in vacuo. The resulting material was purified by flash chromatography on silica gel followed by elution with 5% acetone in n-hexane to give 5 (0.12 mmol, 46 mg) as a white solid.

Sodium borohydride {1.0 mmol, 39.8 mg) was added to a solution of 5 {0.12 mmol, 46 mg) in methanol (10 mL) and the solution was stirred for 15 min. The reaction was stopped with water. The reaction mixture was then diluted with ethyl acetate, and the organic phase was washed with aqueous acid and base. The organic phase was dried and evaporated in vacuo. The resulting material was purified by flash chromatography to give 6 (0.08 mmol, 36 mg) as a white solid.

The spectral data for compound 6 were: H NMR (500 MHz, CDCl 3 ) δ 7.90 (d, 1H), 7.60 (d, 2H), 7.5-7.3 (m, 5H), 6.30 ( d, 2H) 4.5 (s, 2H), 2.3(s, 2H).

Synthesis of p38 inhibitor compound 7

The amine alcohol (500 mg, 1.43 mmol), which was prepared in the same manner as 4, was dissolved in dichloromethane. Triethylamine (433 mg, 4.29 mmol) was added, followed by acetyl chloride (168 mg, 2.15 mmol). The mixture was stirred at room temperature for one hour, poured into water, and extracted with dichloromethane. The organic extract was evaporated in vacuo and the residue was dissolved in 10.0 ml of toluene. A 20% solution of phosgene in toluene (5.0 mL) was added and the solution was refluxed for two hours. The solution was cooled and 5.0 mL of concentrated ammonium hydroxide was added, which precipitated a white solid. The mixture was poured into water and extracted with toluene. The organic extract was dried (MgSO 4 ) and evaporated in vacuo to give 205 mg of the urea acetate 7 as a white solid.

The spectral data for compound 7 were: <1>H NMR (500 MHz, CDCl 3 ) 67.80 (d, 1H), 7.62-7.50 (rn, 2H), 7.25-7.0 (m, 5H ) , 6.59 (d, 1H), 5.1 (s, 2H), 2.12 (s, 3H), HRMS showed MH+ 434.2 as the major peak.

Synthesis of p38 inhibitor compound 8

The urea alcohol (548 mg, 1.4 mmol), which was prepared in the same manner as 6, was dissolved in 5.0 mL of toluene. A 20% solution of phosgene in toluene (5.0 mL) was added and the solution was refluxed for two hours. The solution was cooled and 5.0 mL of concentrated ammonium hydroxide was added, which precipitated a white solid. The mixture was poured into water and extracted with toluene. The organic extract was dried (MgSO 4 ) and evaporated in vacuo to give 284 mg of the carbamate 8 as a white solid.

The spectral data for compound 8 were: <1>H NMR (500 MHz, CDCl 3 δ 7.77 (d, 1H), 7.55-7.45 (m, 2H), 7.15-6.95 (m, 5H ) 6.50 (d, 1H) 5.40 (br s, 2H), 5.00 (s, 2H).HRMS showed MH+ 435.1 as the main peak.

EXAMPLE 2

Synthesis of p38 inhibitor compound 16

One equivalent of 2,6-dichloropyridine-4-carboxylic acid was dissolved in THF. The solution was cooled to 0 °C and 1 equivalent of borane dimethyl sulfide complex was added. The solution was stirred at room temperature for 12 hours. The mixture was poured into water and extracted with diethyl ether. The ether extract was dried and evaporated in vacuo to give 9 in 93% yield.

One equivalent of 9 was dissolved in methylene chloride. One equivalent of methyl chloromethyl ether was added, followed by the addition of one equivalent of ethyldiisopropylamine. The reaction was stirred at room temperature for several hours, poured into water and extracted with a water immiscible solvent. The extract was dried and evaporated in vacuo to give 10 in 86% yield.

One equivalent of potassium t-butoxide was added to a solution of one equivalent of 2,6-dichlorophenylacetonitrile in THF at room temperature. The mixture was stirred at room temperature for 30 minutes, and a solution of dichloropyridine 10 in THF was added. After stirring for 1.5 hours, the mixture was poured into aqueous ammonium chloride and extracted with ethyl acetate. The extract was dried and evaporated in vacuo. The residue was purified by flash chromatography to give 11 in 79% yield as a white powder.

The acetal 11 was mixed with concentrated hydrochloric acid and stirred for several hours. The mixture was extracted with an organic solvent immiscible with water. The extract was washed with saturated aqueous NaHCO 3 , dried, and evaporated in vacuo to give 12.

The nitrile 12 was mixed with concentrated sulfuric acid and heated to 100 °C for several minutes. The mixture was cooled, poured onto ice, and filtered to give 13.

One equivalent of the chloropyridine 13 was dissolved in 1,2-dimetoxyethane. One equivalent of 3-chloro-2-methylphenylboronic acid was added. A solution of one equivalent of sodium carbonate in water was added to a catalytic amount of tetrakis(triphenylphosphine)-palladium (0). The mixture was heated to 80 °C for several hours. The mixture was poured into water and extracted with an organic solvent immiscible with water. The extract was dried, evaporated in vacuo and purified by flash chromatography to give 14.

One equivalent of the alcohol 14 was dissolved in THF. The solution was cooled to 0 °C and one ampoule of methanesulfonyl chloride was added followed by one equivalent of triethylamine. The solution was stirred for several hours, poured into water, and extracted with a solvent that does not mix with water. The extract was dried and evaporated in vacuo to give the crude mesylate 15.

One equivalent of the methanesulfonyl ester 15 was dissolved in THF. The solution was cooled to 0 °C and one equivalent of N-ethyl piperazine was added followed by one equivalent of triethylamine. The solution was stirred for several hours, poured into water and extracted with a solvent immiscible with water. The extract was dried, evaporated and purified by flash chromatography to give the pure amine 16.

The spectral data for compound 16 are: <1>H NMR (500 MHz, CDCl 3 ) δ 9.85 (br s, 1H), 7.47 (dd, 1H), 7.42 (d, 1H), 7.27 ( m, 5H), 6.75 (s, 1H), 5.95 (s, 1H), 5.7 (br S, 1H) 3.5 (Abq, 2H), 2.5-2.3 (m , 10H), 2.3 (s, 3H), 1.2 (t, 3H).

EXAMPLE 2

Cloning of p38-kinase in insect cells

Two splice variants of human p38 kinase, CSBP1 and CSBP2, have been identified. Specific oligonucleotide primers were used to amplify the coding region of CSBP2 cDNA using a HeLa cell library (Stratagen) as a template. The polymerase chain reaction product was cloned into the pET-15b vector (Novagen). The baculovirus transfer vector, pVL-(His)6-p38 was constructed by subcloning an XbaI-BamHI fragment of pET15b-(His)6-p38 into the complementary sites of plasmid pVL1392 (Pharmingen).

Plasmid pVL-(His)6-p38 directed the synthesis of a recombinant protein consisting of a 23-residue peptide

(MGSSHHHHHSSGLVPRGSHMLE, where LVPRGS represents a thrombin cleavage site) fused in reading frame to the N-terminus of p38, as confirmed by DNA sequencing and by N-terminal sequencing of the expressed protein. Monolayer culture of Spodoptera frugiperda-(Sf9) insect cells (ATCC) was maintained in TNM-FH medium (Gibco BRL) supplemented with 10% fetal calf serum in a T-flask at 27°C. Sf9 cells in log phase were co-transfected with linear viral DNA of Autographa califonica nuclear polyhedrosis virus (Pharmingen) and transfer vector pVL-(His)6-p38 using Lipofectin (Invitrogen). The individual recombinant baculovirus clones were purified by plaque assay using 1% low-melting agarose.

EXAMPLE 3

Expression and purification of recombinant p38 kinase

Trichoplusia ni (Tn-368) High-Five™ cells (Invitrogen) were cultured in suspension in Exel-405 protein-free medium (JRH Bioscience) in a shaking flask at 27°C. Cells at a density of 1.5 x 10<6> cells/ml were infected with the recombinant baculovirus described above at a multiplicity of infection of 5. The expression level of recombinant p38 was monitored by immunoblotting using an anti- rabbit p38 antibody (Santa Cruz Biotechnology). The cell mass was harvested 72 hours after infection when the expression level of p38 reached the maximum.

Frozen cell paste from cells expressing (His)6-tagged p38 was thawed in 5 volumes of Buffer A (50 mM Nah2PO4 pH 8.0, 200 mM NaCl, 2 mM p-Mercaptoolethanol, 10% glycerol and 0.2 mM PMSF) . After mechanical disruption of the cells in a microfluidizer, the lysate was centrifuged at 30,000 x g for 30 min. The supernatant was incubated in portions for 3-5 hours at 4°C with Talon™ (Clontech) metal affinity resin at a ratio of 1 ml of resin per 2-4 mg of expected p38. The resin settled by centrifugation at 500 x g for 5 minutes and gently washed in portions with buffer A. The resin was slurried and loaded onto a column (about 2.6 x 5.0 cm) and washed with buffer A + 5 mM imidazole.

(His)e-p38 was eluted with buffer A + 100 mM imidazole and then dialyzed overnight at 4 °C against 2 liters of buffer B, (50 mM HEPES, pH 7.5, 25 mM β-glycerophosphate, 5% glycerin, 2mM DTT). The His6 tag was removed by adding 1.5 units of thrombin (Calbiochem) per mg of p38 and incubating at 20°C for 2-3 hours. The thrombin was stopped by the addition of 0.2 mM PMSF, and then the sample was loaded onto a 2 ml benzamidine-agarose (American International Chemical) column.

The flow-through fraction was directly loaded onto a 2.6 x 5.0 cm Q-Sepharose (Pharmacia) column previously equilibrated in buffer B + 0.2 mM PMSF. P38 was eluted with a 20 column volume linear gradient to 0.6 M NaCl in buffer B. The eluted protein peak was pooled and dialyzed overnight at 4 °C against buffer C (50 mM HEPES pH 7.5, 5% glycerin, 50 mM NaCl, 2 mM DTT, 0.2 mM PMSF).

The dialyzed protein was concentrated in a Centriprep (Amicon) to 3-4 ml and applied to a 2.6 x 100 cm Sepfacryl S-100HR (Pharmacia) column. The protein was eluted at a flow rate of 35 ml/h. The main peak was pooled, adjusted to 20 mM DTT, concentrated to 10-80 mg/ml and frozen in aliquots at -70°C or used immediately.

EXAMPLE 4

Activation of p38

P38 was activated by combining 0.5 mg/ml p38 with 0.005 mg/ml DD double mutant MKK6 in buffer B + 10 mM MgCl 2 , 2 mM ATP, 0.2 mM Na 2 VO 4 , for 30 min at 20°C. The activation mixture was then loaded onto a 1.0 x 10 cmMonoQ column (Pharmacia) and eluted with a linear 20 column volume gradient to 1.0 M NaCl in buffer B. The activated p38 eluted after ADP and ATP. The activated p38 peak was pooled and dialyzed against buffer B + 0.2mM Na 2 VO 4 to remove NaCl. The dialyzed protein was adjusted to 1.1 M potassium phosphate by addition of a 4.0 M stock solution and loaded onto a 1.0 x 10 cm HIC (Rainin Hydropore) column previously equilibrated in buffer D (10% glycerin, 20 mM p- glycerophosphate, 2.0mM DDT) + 1.1MK2HP04. The protein was eluted with a 20 column volume linear gradient to buffer D + 50mM K 2 HPO 4 . The doubly phosphorylated p38 eluted as the major peak and was pooled for dialysis against buffer B + 0.2mM Na 2 VO 4 . The activated p38 was stored at -70°C.

EXAMPLE 5

P38 inhibition assays

A. Inhibition of EGF receptor peptide phosphorylation.

This assay was performed in the presence of 10 mM MgCl 2 , 25 mM β-glycerophosphate, 10% glycerin, and 100 mM HEPES buffer at pH 7.6. For a typical IC 50 determination, a stock solution was prepared containing all the above components and activated p38 (5 nM). The stock solution was aliquoted into vials. A fixed volume of DMSO or inhibitor in DMSO (final concentration of DMSO in the reaction was 5%) was introduced to each vial, mixed and incubated for 15 min at room temperature. EGF receptor peptide, KRELVEPLTPSGEAPNQALLR, a phosphoryl acceptor in p38-catalyzed kinase reaction (1), was added to each vial to a final concentration of 200 µM. The kinase reaction was initiated with ATP (100 µM) and the vials were incubated at 30 °C. After 30 minutes, the reactions were stopped with an equal volume of 10% trifluoroacetic acid (TFA). The phosphorylated peptide was quantified by HPLC analysis. Separation of phosphorylated peptide from the unphosphorylated peptide was achieved on a reversed phase column (deltapak, 5um, C18 100D, Part no. 011795) with a binary gradient of water and acetonitrile, each containing 0.1% TFA. IC 50 (concentration of inhibitor giving 50% inhibition) was determined by plotting percent (%) residual activity against inhibitor concentration.

B. Inhibition of ATPase activity

This assay is performed in the presence of 10 mM MgCl 2 , 25 mM p-glycerophosphate 10% glycerin and 100 mM HEPES buffer at pH 7.6. For a typical Ki determination, the Km for ATP in the ATPase activity of activated p38 reaction is determined in the absence of inhibitor and in the presence of two concentrations of inhibitor. A stock solution is prepared containing all the above components and activated p38 (60 nM). The stock solution is aliquoted into ampoules. A fixed volume of DMSO or inhibitor in DMSO (final concentration of DMSO in reaction was 2.5%) is introduced to each vial, mixed and incubated for 15 minutes at room temperature. The reaction is initiated by adding different concentrations of ATP and then incubated at 30°C. After 30 minutes, the reactions are stopped with 50 µM EDTA (0.1 M, final concentration), pH 8.0. The product of p38 ATPase activity, ADP, is quantified by HPLC analyses.

Separation of ADP from ATP is achieved on a reversed phase column (Supelcosil, LC-18, 3 µm, part no. 5-8985) using a binary solvent gradient of the following composition: Solvent A - 0.1 M phosphate buffer containing 8 mM tetrabutylammonium hydrogen sulfate (Sigma Chemical Co., catalog no. T-7158), Solvent B - Solvent A with 30% methanol.

Ki is determined from velocity data as a function of inhibitor and ATP concentrations.

P38 inhibitors according to the present invention will inhibit the ATPase activity of p38.

C. Inhibition of IL-1, TNF, IL-6 and IL-8

Production in LPS-stimulated PBMCs

Inhibitors were serially diluted in DMSO from a 20 mM stock solution. At least 6 serial dilutions were prepared. Then 4x inhibitor stock solutions were prepared by adding 4 µl of an inhibitor dilution to 1 ml of RPMI 1640 medium/10% fetal calf serum. The 4x inhibitor stock solutions contained inhibitor at concentrations of 80 µM, 32 µM, 12.8 µM, 5.12 µM, 2.048 µM, 0.819 µM, 0.328 µM, 0.131 µM, 0.052 µM, 0.021 µM, etc. The 4x inhibitor stock solutions were prewarmed at 37 °C until use.

"Buffy cells" from fresh human blood were separated from other cells in a Vacutainer CPT from Becton & Dickinson (containing 4 ml of blood and sufficient DPBS without Mg<2+>/Ca<2+ >to fill the tube) by centrifugation at 1500 x g for 15 minutes. Peripheral blood mononuclear cells (PBMCs), located at the top of the gradient in the Vacutainer, were removed and washed twice with RPMI 1640 medium/10%

fetal calf serum. PBMCs were collected by centrifugation at 500 x g for 10 min. The total cell number was determined using a Neubauer cell chamber and the cells were adjusted to a concentration of 4.8 x 10<6> cells/ml in cell culture medium (RPMI 1640 supplemented with 10% fetal calf serum).

Alternatively, whole blood containing an anticoagulant was used directly in the assay.

100 µl of cell suspension or whole blood was placed in each well of a 96-well cell culture plate. Then 50 µl of the 4x inhibitor stock solution was added to the cells. Finally, 50 µl of a lipopolysaccharide (LPS) working stock solution (16 ng/ml in cell culture medium) was added to give a final concentration of 4 ng/ml LPS in the assay. The total assay volume in the vehicle control was also adjusted to 200 µl by adding 50 µl of cell culture medium. The PBMCs or whole blood were then incubated overnight (for 12-15 hours) at 37°C/5% CO 2 in a humidified atmosphere.

The next day, the cells were mixed on a shaker for 3-5 min before centrifugation at 500 x g for 5 min. Cell culture supernatants were harvested and analyzed by ELISA for levels of IL-1b (R & D Systems, Quantikine kits, #DBL50), TNF-oc (BioSource, #KHC3012), IL-6 (Endogen, #EH2-IL6) and IL- 8 (Endogen, #EH2-IL8) according to the manufacturer's instructions. The ELISA data are used to generate dose-response curves from which IC50 values are derived.

Results for the kinase assay ("kinase"; subsection A, above), IL-1 and TNF in LPS-stimulated PBMCs ("cell") and IL-1, TNF and IL-6 in whole blood ("WB") for various p38 inhibitors according to the present invention is shown in table 7 below: Other p38 inhibitors according to the present invention will also inhibit phosphorylation of EGF receptor peptide and will inhibit the production of IL-1, TNF and IL-6 in addition to IL-8 in LPS-stimulated PBMCs or in whole blood.

D. Inhibition of IL-6 and IL-8

Production in IL-1-stimulated PBMCs

This assay is performed on PBMCs exactly as above except that 50 µl of an IL-1b working stock solution (2 ng/ml in cell culture medium) is added to the assay instead of the (LPS) working stock solution.

Cell culture supernatants are harvested as described above and analyzed by ELISA for levels of IL-6 (Endogen, #EH2-IL6) and IL-8 (Endogen, #EH2-IL8) according to the manufacturer's instructions. The ELISA data are used to generate dose-response curves from which IC 50 values are derived.

E. Inhibition of LPS-induced

prostaglandin endoperoxide synthase-2

(PGHS-2, or COX-2) induction in PBMCs

Human peripheral mononuclear cells (PBMCs) are isolated from buffy coats of fresh human blood by centrifugation in a Vacutainer CPT (Becton & Dickinson). 15 x 10<6> cells are seeded in a 6-well tissue culture plate containing RPMI 164 0 supplemented with 10% fetal calf serum, 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine. Compounds are added to 0.2, 2.0 and 20 µM final concentration in DMSO. LPS is then added at a final concentration of 4 ng/ml to induce enzyme expression. The final culture volume is 10 ml/well.

After overnight incubation at 37°C, 5% CO2, the cells are harvested by scraping and subsequent centrifugation, the supernatant is removed, and the cells are washed twice in ice-cold DPBS (Dulbecco's phosphate-buffered saline, BioWhittaker). The cells are lysed on ice for 10 minutes in 50 µl of cold lysis buffer. (20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% Triton-X-100, 1% deoxycholic acid, 0.1% SDS, 1 mM EDTA, 2% aprotinin (Sigma), 10 µg/ml pepstatin , 1 mM DTT) containing 2 mM PMSF, 1 mM benzamidine, 1 mM DTT) containing 1 µl Benzonase (DNAse from Merck). The protein concentration in each sample is determined using the BCA assay (Pierce) and bovine serum albumin as a standard. Then the protein concentration for each sample is adjusted to 1 mg/ml in cold lysis buffer. 100 µl of lysate is added to an equal volume of 2xSDS AGE loading buffer, and the sample is boiled for 5 minutes. Proteins (30 µg/lane) are fractionated by size on 4-20% SDS PAGE gradient gels (Novex) and then transferred onto nitrocellulose membrane by electrophoretic methods for 2 h at 100 mA in Towbin transfer buffer (25 mM Tris, 192 mM glycine) containing 20% methanol. After transfer, the membrane is pretreated for 1 hour at room temperature with blocking buffer (5% nonfat dry milk in DPBS supplemented with 0.1% Tween-20) and washed 3 times in DPBS/0.1% Tween-20. The membrane is incubated overnight at 4°C with a 1:250 dilution of monoclonal anti-COX-2 antibody (Transduction Laboratories) in blocking buffer. After 3 washes in DPBS/0.1% Tween-20, the membrane is incubated with a 1:1000 dilution of horseradish peroxidase-conjugated sheep antiserum to mouse Ig (Amersham) in oxygen blocking buffer at room temperature. Then the membrane is again washed 3 times in DPBS/0.1% Tween-20. An ECL detection system (SuperSignal™ CL-HRP Substrate System, Pierce) is used to determine the expression levels of COX-2.

While we previously presented herein a number of embodiments of the present invention, it is obvious that our basic construction can be modified to provide other embodiments that employ the methods of the present invention.

Claims (35)

1. Compound with formula: wherein each Q 1 and Q 2 is independently selected from phenyl or naphthyl; the rings constituting Qi are substituted with 1 to 4 substituents each of which is independently selected from halo; C1-C3-alkyl or O-(C1-C3)-alkyl; the rings constituting Q2 are optionally substituted with up to 4 substituents, each of which is independently selected from halo; straight or branched C 1 -C 3 alkyl optionally substituted with OH; R' is selected from hydrogen; (C 1 -C 3 )-alkyl; phenyl or phenyl substituted with 1 to 3 substituents independently selected from halo, methoxy, methyl or ethyl; or a 5-6 membered heterocyclic ring system optionally substituted with 1-3 substituents independently selected from halo, methoxy, hydroxy, methyl or ethyl, wherein the heterocyclic ring system is selected from pyridyl, morpholinyl, piperazinyl, dihydrothiazolyl, tetrazolyl, piperidinyl, triazolyl or pyrrolidinyl ; R 3 is selected from a 5-8 membered aromatic or non-aromatic carbocyclic or heterocyclic ring system, each optionally substituted with R', R 4 or -C(0)R', wherein the ring system is selected from phenyl, cyclohexyl, piperazinyl, pyrrolidinyl , piperidinyl, homopiperazinyl, morpholinyl, imidazolyl, or triazolyl; R<4> is straight or branched {C1-C4) alkyl optionally substituted with OR', CO2R' or CON(R')2; or a 5-6 membered carbocyclic or heterocyclic ring system; R<5> is selected from hydrogen; (C 1 -C 3 )-alkyl, pyridinyl, morpholinyl, or tetrazolyl; W is selected from H; N(R2)C(O)-OR2; N(R2)C(O)-N(R2)2; N(R<2>)C{0)-N(R<2>)(R<3>); N(R<2>)C(O)-R<2>; N(R<2>)2; C(O)-R 2 ; C(O)-N{R 2 ) 2 ; C(0)-OR<2>; J; or straight or branched (C1-C4) alkyl optionally substituted with N{R')2, OR', CO2R', CON{R')2, R<3>, SO2N(R<2>)2, 0C(0)R2, 0C(0)R', OC(0)N{R<2>)2, -N(R<4>){R<5>), -C (0) N (R5) (R2) , -N (R2) C (0) N (R2) (R<5>), -NC(0)0R<5>, -0C (0) N{R2) (R5) , -J; NHS02-R2, NHS02-R<3>, or 0P03H2; provided that if U is W, W is not R<3->substituted C 1 -alkyl; each R is independently selected from hydrogen, -R<2>, -N(R<2>)2 or -OR<2>; R<2> is selected from hydrogen or (C1-C3)-alkyl, optionally substituted with -N {R' ) 2 , -OR' , -C (0)-N (R') 2 , -C<0) -OR', -C(O)N=C(NH)2 or R<3>; Y is C; Z is CH or N; U is selected from R or W; V is -C(O)NH 2 ; A, B and C are independently selected from -O-, -NH- or -CH2-J is a straight-chain or branched (C1-C4)-alkyl derivative substituted with 1-3 substituents selected from D, -T-C(0) R' , or -PO 3 H 2 ; D is selected from the group T is either 0 or NH; and G is either NH2 or OH, provided that in compounds of formula (Ia) where W is hydrogen, then U is not hydrogen, and in compounds of formula (Ia) where W is hydrogen and U is -R<2>, -N(R<2>)2 or -OR2, then each of these R2 is not hydrogen, (C1-C3)-alkyl or (Ci-C3) -alkyl substituted by -N(R')2, -OR', -C (O) -N (R' ) 2, -C(0)-0R' or an unsubstituted 5-6- linked aromatic carbocyclic or heterocyclic ring system. 2. Compound according to claim 1, wherein Qi is a phenyl containing 1 to 3 substituents independently selected from chlorine, fluorine, bromine, -CH3, -OCH3 or -0(CH2)2CH3 and wherein at least one of said substituents is in ortho- the position. 3. A compound according to claim 2, wherein Qi contains at least two substituents which are both in the ortho position. 4. The compound of claim 2, wherein Qi is selected from: 5. A compound according to claim 4, wherein Qi is selected from 2,6-difluorophenyl or 2,6-dichlorophenyl. 6. A compound according to claim 1, wherein Q2 is selected from phenyl or naphthyl and wherein Q2 optionally contains up to 3 substituents, each of which is independently selected from chlorine, fluorine, bromine, methyl, ethyl, isopropyl, -OCH3 and -CH2OH. 7. A compound according to claim 6, wherein Q2 is selected from: or unsubstituted phenyl. 8. A compound according to claim 7, wherein Q2 is selected from phenyl, 2-isopropylphenyl, 3,4-dimethylphenyl, 2-ethylphenyl, 3-fluorophenyl, 2-methylphenyl, 3-chloro-4-fluorophenyl, 3-chlorophenyl, 2- methyl-4-chlorophenyl, 2-bromophenyl, 2-methylenehydroxyphenyl, 4-fluorophenyl, 2-methyl-4-fluorophenyl, 2-chloro-4-fluorophenyl, 2,4-difluorophenyl, 2-methylenehydroxy-4-fluorophenyl, 1-naphthyl, 3-chloro-2-methylenehydroxy, 3-chloro-2-methyl or 4-fluoro-2-methyl. 9. A compound according to claim 1, wherein each R and U bonded to Y is independently selected from hydrogen or methyl. 10. A compound according to claim 1, wherein U, W or both U and W are a 0-4 atom chain terminating in an alcohol, amine, carboxylic acid, ester, amide or heterocycle. 11. Compound according to claim 10, wherein U, W or both U and W are selected from: 12. Compound according to claim 11, wherein U, W, or both U and W are selected from: 13. A compound according to claim 1, wherein the compound is selected from any of the following compounds: 14. A compound according to claim 1, wherein the compound is and X is selected from H, or 15. A compound according to claim 1, wherein the compound is and X is selected from NH 2 or N{CH 3 ) 2 . 16. A compound according to claim 1, wherein the compound is and X is selected from OH, NH 2 , or N(CH 3 ) 2 . 17. A compound according to claim 1, wherein the compound is and X is selected from OH, NH 2 , or N(CH 3 ) 2 . 18. A compound according to claim 1, wherein the compound is and X is selected from OH, NH2, N(CH3)2, 19. A compound according to claim 1, wherein the compound is where X = H, 20. A compound according to claim 1, wherein the compound is wherein X = 21. A compound according to claim 1, wherein the compound is wherein X is 22. A compound according to claim 1, wherein the compound is selected from any of 23. A pharmaceutical composition comprising an amount of a compound according to any one of claims 1 to 22 effective to inhibit p38 and a pharmaceutically acceptable carrier. 24. Use of a compound according to any one of claims 1-23 for the preparation of a drug to treat or prevent inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative disorders, infectious diseases, neurodegenerative diseases, allergies, reperfusion/ischemia in stroke, heart - attack, angiogenic disorders, organ hypoxia, vascular hyperplasia, cardiac hypertrophy, thrombin-induced platelet aggregation or conditions associated with prostaglandin endoperoxide synthase-2 in a patient. 25. Use according to claim 24, in which the drug is for the treatment or prevention of an inflammatory disease selected from acute pancreatitis, chronic pancreatitis, asthma, allergies, or shock lung (ARDS, adult respiratory distress syndrome). 26. Use according to claim 24, wherein the drug is for the treatment or prevention of an autoimmune disease selected from glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia , atopic dermatitis, chronic active hepatitis, myasthenia gravis, multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, psoriasis, or graft-versus-host reaction. 27. Use according to claim 24, wherein the drug is for the treatment or prevention of a destructive bone disorder selected from osteoarthritis, osteoporosis or multiple myeloma-related bone disorder. 28. Use according to claim 24, wherein the drug is for the treatment or prevention of a proliferative disease selected from acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, or multiple myeloma. 29. Use according to claim 24, wherein the drug is for the treatment or prevention of an infectious disease selected from sepsis, septic shock or shigellosis. 30. Use according to claim 24, in which the drug is for the treatment or prevention of a viral disease selected from acute hepatitis infection, HIV infection or CMV rhinitis. 31. Use according to claim 24, wherein the drug is for the treatment or prevention of a neurodegenerative disease selected from Alzheimer's disease, Parkinson's disease, cerebral ischemia or neurodegenerative disease caused by traumatic injury. 32. Use according to claim 24, in which the drug is for the treatment or prevention of ischemia/reperfusion in stroke or myocardial ischemia, renal ischemia, heart attack, organ hypoxia or thrombin-induced platelet aggregation. 33. Use according to claim 24, in which the drug is for the treatment or prevention of a condition associated with prostaglandin edoperoxide synthase-2 selected from oedema, fever, analgesia or pain. 34. Use according to claim 33, wherein said pain is selected from neuromuscular pain, headache, cancer pain, toothache or arthritis pain. 35. Use according to claim 24, wherein the drug is for the treatment or prevention of an angiogenic disorder selected from hard tumors, ocular neovascularization, or infantile hemangioma.